The regulatory response of cells to temperature change is highly conserved among all organisms. Our long term goal is to understand the function and regulation of this cellular response by investigating it in E. coli, which contains two groups of heat regulated genes, one transcribed by sigma32 and the other transcribed by sigmaE each transcribed by a different alternative sigma factor. In general, these genes encode heat shock proteins (hsps) that are proteases or chaperons and function to maintain the folding state of the cytoplasmic compartment of the cell. In the current granting period, our specific aims are to: 1. Define the role of the ATPase cycle and binding to DnaJ in the chaperone function of DnaK by selecting mutants defective in these activities and characterizing them genetically and functionally. 2. Examine the interaction of K,J, and E with the lambdaP protein by using environmentally sensitive fluorescent probes and mutant analysis to investigate how interaction of K,J, and E with lambdaP alters its folded state and to identify the sites on lambdaP that interact with K and J. 3. Examine the interaction of K,J, E with sigma32, by determining binding constants for interaction, measuring how K,J, and E binding affect the interaction between sigma32,and core RNA polymerase using a solution based competition assay and characterizing sigma32, mutants refractory to inactivation. 4. Examine the degradation of sigma32 by setting up an in vitro system that degrades sigma32 and examining the roles of the HflB, K,J, and E hsps in this process. 5. Investigate the regulation of sigmaE by determining the signal transduction pathway that links events outside the cytoplasm to sigmaE activity, and then identify members of the sigmaE regulon. In addition to providing fundamental knowledge on hsp function and regulation, these studies have direct medical relevance. T cells reactive to heat shock proteins may provide a first line of defense against infection. In addition, studies on thermotolerance are important in determining the effectiveness of heat treatment in some cancer therapies.